The invention provides genetically engineered microorganisms with modified hydrogenase activity and methods related thereto. Typically, the microorganisms are C1-fixing microorganisms with one or more disruptive mutations in a hydrogenase enzyme or a hydrogenase accessory enzyme. The microorganisms may have improved tolerance to toxins, such as acetylene, isocyanide, ammonium, or nitric oxide, improved production of products, such as ethanol, 2,3-butanediol, and isopropanol, and/or improved fixation of carbon, such as carbon derived from CO or CO.sub.2.

Compositions and methods are provided for an O.sub.2 tolerant FeFe hydrogenase. The hydrogenases of the invention comprise specific amino acid substitutions relative to the native, or wild-type enzymes.